Enhanced production of hyperthermophilic Pyrococcus furiosus β-glucosidase in Corynebacterium glutamicum by optimization of the promoter, vector backbone, and His-tag location

Abstract

A hyperthermophilic β-glucosidase from Pyrococcus furiosus (PfBGL) is a highly stable and active glycoside hydrolase, well-suited for a wide range of applications. Although PfBGL has been successfully expressed in Escherichia coli, the use of this host limits its applicability in the healthcare and food processing industries due to safety concerns associated with E. coli-based expression systems. Recently, Corynebacterium glutamicum has emerged as a safe and versatile microbial platform for the expression of recombinant proteins used in food processing, pharmaceutical development, therapeutic enzyme production, and probiotic applications. Despite these advantages, heterologous expression in C. glutamicum is often hindered by low protein yields, and PfBGL expression in this host has not been previously explored. In this study, we report for the first time the production of PfBGL in C. glutamicum, achieving a 15-fold enhancement through the optimization of the promoter, the vector backbone, and the His-tag location. Among four different promoters, the P_trc promoter with RBS_T7 yielded the highest PfBGL expression. For the P_trc-RBS_T7 combination, the PfBGL expression levels further varied depending on the vector backbone. Interestingly, placing the His-tag at the N-terminus of PfBGL not only increased its expression in C. glutamicum, but also enhanced its enzymatic activity (k_cat/K_m), when compared to C-terminal tagging. Overall, this study showcases a simple yet effective strategy at both genetic and protein levels to enhance PfBGL production in C. glutamicum, thereby broadening its utility as a host for diverse protein production applications.